| Abstract | Methylmalonyl-CoA mutase (MCM, E.C. 5.4.99.2), a coenzyme B12-dependent enzyme, catalyses the inter conversion of succinyl-CoA and methylmalonyl-CoA. The gene (sbm) encoding this enzyme is found in Escherichia coli (E. coli) at 62.3min on the E. coli chromosome. However, the metabolic role of this enzyme in the organism is not known. This project involves an investigation into this metabolic obscurity.
The sbm gene is part of a four gene operon which also includes argK (or ygfD) that codes for a protein kinase catalysing the phosphorylation of two periplasmic binding proteins involved in cationic amino acid transport, ygfG that codes for methylmalonyl-CoA decarboxylase and ygfH that codes for propionyl-CoA: succinyl-CoA transferase. From existing literature we suspect that this operon, including the sbm gene, could be involved in the utilisation of unusual carbon
sources such as succinate and propionate. An insertion mutant of the sbm gene created by transposon mediated mutagenesis was used for investigating the role of this gene. The wild type E. coli K12 strain, E. coli TR6524 and the mutant E.
coli K12 (sbm::MudJ) were used in this study.
Growth of the two strains (E. coli TR6524 and FA1P1) in minimal media with three different concentrations (0.05, 0.5, 5.0μg/mL) of vitamin B12 and in the presence succinate, propionate or glucose as the sole source of carbon, was
studied. Growth was typical in media with glucose with no major differences in the growth pattern of the wild type and mutant strain. However, the two strains exhibited a differential growth pattern in media containing succinate, with the wild type growing faster than the mutant, indicating the role of the sbm gene in the utilisation of this carbon source. Growth in media containing propionate as the sole carbon source indicated only marginal differences in the growth pattern of the wild type and mutant strain. This result possibly suggests that the other pathways for propionate utilisation in E. coli compensate for the lack of a functional Sbm protein in the mutant strain. Promoter analysis indicated the presence of a promoter induced by σS, a transcription factor involved in the expression of proteins under stress or stationary phase growth conditions. Reverse transcription polymerase chain reaction (RT-PCR) studies of the genes of the sbm operon (sbm-argK-ygfGygfH) under the same growth conditions were carried out. Densitometric analysis of the PCR products suggested that the transcription level of sbm was higher in E. coli grown in succinate as compared to when grown in glucose and not as much when grown in propionate indicating a transcriptional level control of the sbm gene expression during the utilisation of succinate. RT-PCR studies also indicated a higher level of transcription of the gene in the stationary phase of the culture during the utilisation of succinate. Real time reverse transcription PCR (QPCR) analysis was used for the absolute quantification of the transcription of the genes of the sbm operon. An increase in the mRNA levels corresponding to the sbm, argK and ygfG genes was observed as E. coli TR6524 growth reached stationary phase, in the presence of succinate or propionate as the sole source of carbon as compared to glucose, In contrast, the highest mRNA levels corresponding to the ygfH gene were observed in the early log-phase of growth.
This indicated a differential transcriptional level control of the genes within the operon. This study further established the possible role of this operon in the utilisation of succinate and propionate.
The MCM enzyme activity measurement in the whole cell extracts of the wild type E. coli K12, grown under the above mentioned conditions, led to the first ever measurement of MCM activity in wild type E. coli. These measurements also revealed a four fold increase of the MCM specific activity in the case of growth in succinate (4.76x10-3U/mg) and a two fold increase for growth in propionate (2.79x10-3U/mg) compared to that observed with growth in glucose (1.37x10-3U/mg), indicating a significant level of involvement of the enzyme in succinate utilisation, and to a lesser extent in propionate utilisation.
The proteomic analysis to understand the gene expression pattern of E. coli TR6524 was carried out using cells harvested at the stationary phase. The results showed that growth conditions induced the expression of transport related (HisJ, DppA) and energy generating proteins (PckA, AceF) required by E. coli to cope with the stressful growth conditions. However, Sbm was not identified among the limited protein spots that were analysed.
Finally, E. coli K12 sbm gene was successfully cloned into B. cereus SPV leading to the development of a metabolically engineered polyhydroxyalkanoate producing strain of B. cereus. The intention was to provide the bacteria with a
natural intracellular source of propionyl-CoA, leading to the production of the P(3HB-co-3HV) copolymer from structurally non related carbon sources like glucose.
Hence, this work has initiated investigation into the metabolic role of the sbm gene product in E. coli. In addition, it has also led to the use of this gene product in metabolic engineering applications. |
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